Project Abstract Breast cancer remains the second-leading cause of cancer-related death in the US. Obesity is an established risk factor for several aggressive breast cancer subtypes, and is also associated with increased breast cancer metastasis and mortality. On a cellular level, obesity drives increased inflammation and dysregulated leptin signaling, both of which have been independently shown to increase cancer cell migration and invasion. However, the exact mechanisms by which these drivers link obesity to increased breast cancer metastasis are poorly understood. Since the prevalence of obesity in the US and many other countries is very high, and the vast majority of breast cancer-related deaths are due to metastatic disease, intervention strategies for breaking the link between obesity and breast cancer metastasis are urgently needed to reduce breast cancer mortality. Several series of metastatic breast cancer cell lines (each series driven by a different genetic alteration and varying in metastatic potential) have been developed that can be used in orthotopic transplant models to study the effects of obesity on metastatic breast cancer. Our preliminary findings in one of these models support the obesity-metastasis link and suggest obesity-induced inflammation may underlie this link. This proposal will use murine models of obesity and metastatic mammary cancer in concert with several state-of-the-art mechanistic approaches, including multiplexed assays of inflammatory markers and a high-throughput single-cell RNA sequencing approach for assessing the tumor microenvironment, to rigorously test the hypothesis that obesity- associated inflammation and leptin signaling act together to enhance breast cancer metastasis by driving a cancer stem-cell phenotype. This hypothesis will be tested with two integrated specific aims: Aim 1. Quantify the impact of obesity and anti-inflammatory treatment on tumor growth, metastasis, metabolic phenotype, and inflammatory and stem-like markers in three separate metastatic murine mammary tumor lines. Aim 2. Define the impact of obesity and anti-inflammatory treatment on mammary tumor gene expression and metastatic potential using a high-throughput, unbiased single-cell RNA-sequencing approach. Using this data, potential drivers in one metastatic cell line will be identified, manipulated using CRISPR, confirmed in vitro and validated in the other two cell lines. Following validation, the altered cell line will be assayed in vivo to test the metastatic potential manipulated line in a pilot mouse study comparing lean and obese animals. This proposal aims to define molecular characteristics enriched in breast cancer metastasis and ultimately to identify therapeutic targets to help decrease breast cancer mortality by limiting metastasis, particularly in the obese patient population. Combined with the exceptional training environment at UNC, comprehensive mentoring from Drs. Hursting and Anders, and a focused training plan, this fellowship will provide a critical foundation for developing my future career as an independently-funded physician-scientist in the field of cancer metastasis.